Bacteriocin piscicolin 126

ABSTRACT

The present invention provides a bacteriocin having a molecular weight of about 4.4 kDa and methods involving the use of this bacteriocin. The bacteriocin preferably has an amino acid sequence as shown in SEQ ID NO:5 (FIG. 3). The present invention also provides a DNA molecule encoding this bacteriocin.

This invention relates to novel bacteriocin molecules havingantimicrobial activity. In one particular application, the bacteriocinaccording to the invention is used as a food preservative.

BACKGROUND OF THE INVENTION

There is a continual need for new food preservatives bearing new anduseful properties. Further, there is growing interest in replacingtraditional "chemical" food preservatives with effective "natural"preservatives, especially those which are specific for pathogenicmicroorganisms and do not harm beneficial food-producing strains. Inthis regard, considerable research has been conducted on bacterialpeptides known as bacteriocins which are often heat stable and haveantimicrobial activity. Two such bacteriocins which are commerciallyproduced for use as food preservatives are nisin and pediocin PA-1.Nisin has been given the status of Generally Regarded as Safe for humanconsumption (GRAS) by the United States FDA. Nisin and pediocin PA-1however, have broad spectrum activity, affecting not only pathogenic,but also beneficial (in the food system) microorganisms.

SUMMARY OF THE INVENTION

Novel bacteriocins having a range of activity that is different to andpreferably narrower than those of nisin and pediocin PA-1 would bedesirable. The present inventors have now identified and isolated a newbacteriocin which has a limited range of activity in comparison to, forexample, nisin and pediocin PA-1.

Thus, in a first aspect, the present invention consists in asubstantially pure preparation of a bacteriocin having a molecular massof about 4.4 kDa and antimicrobial activity substantially according toTable 2.

In a preferred embodiment of the present invention the bacteriocin hasan amino acid sequence substantially identical to the sequence shown inFIG. 3.

In a second aspect the present invention consists in a biologically pureculture of bacterial strain JG126.

A sample of bacterial strain JG126 was deposited under the BudapestTreaty with Australian Government Analytical Laboratories (AGAL), 1Saukin Street, Pymble, New South Wales, Australia, on Dec. 21 1994, andwas accorded accession No. N94/61478.

In a third aspect the present invention consists in an isolated DNAmolecule encoding the bacteriocin of the first aspect of the presentinvention.

In a preferred embodiment of this aspect of the present invention theDNA molecule includes a nucleotide sequence as shown in FIG. 6.

In a fourth aspect the invention consists in a method of preservingfoods or beverages, the method comprising adding to the food or beveragean effective antimicrobial amount of a bacteriocin of the first aspectof the present invention.

The bacteriocin of the present invention may be isolated from naturalsources, or produced by recombinant DNA techniques well known in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following non-limiting examples and the accompanying figures.

Figure Legends

FIGS. 1A-1C: Purification of piscicolin 126 by reversed-phase HPLC

(A) C18 reversed-phase HPLC separation of piscicolin 126 on a 4.6×250 mmSpherisorb ODS II (80 Å pore size, 5 μm particle size) column developedwith the gradient of increasing acetonitrile concentration indicated bythe dashed line. The piscicolin 126 had been previously partiallypurified by ion-exchange chromatography (not shown). The peak indicatedby the arrow was collected and shown to have antimicrobial activity. (B)Rechromatography by C18 reversed-phase HPLC of the peak of piscicolin126 collected as described in panel A. The column (2.1×250 mm SpherisorbODS II [80 Å pore size, 5 μm particle size]) was developed with thegradient of increasing acetonitrile concentration indicated by thedashed line. (C) Electropherogram of purified piscicolin 126 separatedby CZE through a 50 μm×50 cm uncoated silica capillary in 20 mM sodiumcitrate (pH 2.5) at 20 kV.

FIG. 2. MALDI-TOF mass spectrum of purified piscicolin 126.

A sample of purified piscicolin 126 was co-analysed along with thebacteriocin SA-FF22 (m=2794.3) and porcine insulin (m=5777.6) and theinstrument was calibrated accordingly. From 36 mass spectra recorded, amass estimate of m=4416.6 ±1.9 was calculated for piscicolin 126.

FIG. 3. Amino Acid Sequence of Piscicolin 126.

FIGS. 4A-C: Analysis of the products formed following treatment ofpiscicolin 126 with the alkylating agent 4-vinylpyridine in either thepresence or absence of a reducing agent.

FIG. 4A: Reversed-phase HPLC separation of piscicolin 126 followingtreatment with 4-vinylpyridine in the (A) absence of and (B) presence ofthe reducing agent 2-mercaptoethanol. The collected peaks from eachseparation were analysed by MALDI-TOF mass spectrometry.

FIG. 4B: (C) peak material from A and

FIG. 4C: D peak material from B. No mass increase was observed in A(sample without reducing agent) suggesting that piscicolin 126 does notcontain free sulphydryl groups while the mass increase in D (210 Da)corresponds to bi-pyridethylation and suggests the piscicolin 126contains a disulphide bridge between the two cysteine residues.

FIG. 5. DNA sequence of the 607 bp Hinf I/Sau 3A DNA fragment encodingthe structural gene for Piscicolin 126.

FIG. 6. DNA sequence and translation of gene encoding Piscicolin 126 andits putative signal peptide.

EXAMPLES

The invention will now be described in more detail with reference to thefollowing examples.

Example 1

Isolation and Characterisation of Bacteriocin-Producing Strain JG126

A number of Gram-positive bacteria were collected from a wide range ofsources and assessed for antimicrobial activity. One strain, JG126, wasisolated from ham and found to be a catalase negative, Gram-positiverod.

To establish the identity of this organism, 16S rDNA was amplified bythe polymerase chain reaction (PCR) from chromosomal DNA of theproducing organism using the primers 5'GAGTTTGATCCTGGCTCAG and5'TACAAGGCCCGGGAACG, corresponding to nucleotides 9-27 and 1394-1378,respectively, in the Eschericia coli 16S rRNA numbering system. Apartial nucleotide sequence of the PCR product was determined usingprimer 9-27 and the sequence compared to all 16S rRNA sequences in theribosomal database of Australian National Genomic Information Service(ANGIS) using the program FastA. The nucleotide sequence was 100%identical to Carnobacterium piscicola, accession no. M58812 in GenBankand it was concluded that JG126 was a strain of C. piscicola.

A sample of this bacterium was deposited under the Budapest Treaty withAustralian Government Analytical Laboratories (AGAL), 1 Saukin Street,Pymble, New South Wales, Australia, on Dec. 21, 1994, and was accordedaccession No. N94/61478.

The activity of the bacteriocin (designated herein as piscicolin 126)was determined as follows by bioassay with Listeria monocytogenes 4A.Bacteriocin was serially diluted with sterile distilled water andaliquots of each dilution were applied to an MRS (de Man, Rogasa andSharpe medium) agar plate and overlaid with 7 ml TSBYE (Tryptohe Soybroth supplemented with 0.6% (w/v) yeast extract) soft agar (0.6% (w/v))seeded with 10⁶ cfu ml⁻¹ of Listeria monocytogenes 4A and incubated at30° C. for 16 h. The activity of piscicolin 126 was expressed inArbitrary Units (AU), defined as the reciprocal of the titre of thehighest dilution demonstrating a detectable zone of growth inhibition inthe indicator lawn culture.

Example 2

Purification of Piscicolin 126

Piscicolin 126 was purified in the following manner:

Overnight cultures of bacterial strain JG126 in acetate-free MRS grownat 22° C. were clarified by centrifugation.

The peptide was precipitated from the culture supernatant by theaddition of ammonium sulphate to 80% saturation and the precipitate wasrecovered by centrifugation.

Peptide was purified by chromatography:

1. by ion exchange chromatography on CM-Sepharose Fast Flow; then

2. by semi analytical Reversed-phase HPLC on Spherisorb ODSII 4.6 ×250mm, 80 Å, 5 mm particle (FIG. 1A); then

3. by analytical reversed-phase HPLC on Spherisorb ODSII 2.1×250 mm, 80Å, 5 mm particle (FIG. 1B).

This resulted in a single peak of peptide (FIG. 1C).

Example 3

Characterisation of the Properties of Piscicolin 126.

The molecular weight (M_(r)) of piscicolin 126 was determined byMALDI-TOF mass spectrum analysis (FIG. 2). The peak of the piscicolin126 corresponds to an M_(r) of 4416.6±1.9 Da.

Piscicolin 126 was digested with a variety of enzymes, including somewith proteolytic activity, and the digests were assessed forantimicrobial activity. The results are presented in Table 1 andindicate that the piscicolin 126 contains peptide material.

Table 1. Enzyme Inactivation of Piscicolin 126

20 μl enzyme solution (200 μg/ml in 0.2 M phosphate buffer, pH 7.0) wasadded to 200 μl of filter sterilised bacteriocin preparation. andincubated at 37° C. for 2 h. Residual bacteriocin activity (ArbitraryUnits ml⁻¹) was determined by bioassay with Listeria monocytogenes 4A.

    ______________________________________                                                        Activity                                                      Enzyme Treatment  AU ml.sup.-1                                                                           %.sup.1                                            ______________________________________                                        Control           12800    100                                                Catalase          12800    100                                                α-Chymotrypsin                                                                            0        0                                                  β-Chymotrypsin                                                                             0        0                                                  Lipase            12800    100                                                Lysozyme          12800    100                                                Pepsin            12800    100                                                Protease I        0        0                                                  Protease XIV      400      3                                                  Protease XXIII    0        0                                                  Trypsin           800      6                                                  ______________________________________                                         .sup.1 % refers to residual bacteriocin activity (following treatment) as     a % of original activity, i.e., unaffected = 100%. completely destroyed =     0%.                                                                      

Example 4

Determination of the Structure of Piscicolin 126

The amino acid sequence of the piscicolin 126 was determined. Thesequence is presented in FIG. 3.

The presence or absence of free sulphydryl groups in piscicolin 126 wasdetermined by analysis of the products formed following treatment ofpiscicolin 126 with the alkylating agent 4-vinylpyridine in either thepresence or absence of the reducing agent 2-mercaptoethanol (FIG. 4). Nomass increase was observed for the sample that had been reacted with4-vinylpyridine in the absence of reducing agent FIG. 4C), indicatingthat piscicolin 126 does not contain free sulphydryl groups. The massincrease of 210 Da observed for the sample reacted with 4-vinylpyrridinein the presence of the reducing agent (FIG. 4D) corresponds tobipyridethylation and indicates that piscicolin 126 contains adisulphide bridge between two cysteine residues.

Example 5

Isolation and characterisation of the Gene Encoding Piscicolin 126

High molecular weight total DNA was isolated from bacterial strain JG126and partially digested with restriction endonuclease Sau3AI. Thecohesive ends were partially filled using Klenow DNA polymerase, dGTPand dATP and ligated into the partially filled Xho I sites of the armsof the LambdaGEM®-12 vector. The ligated DNA was packaged into phagesand used to transfect E.coli strain LE392.

A piscicolin 126 specific gene probe was prepared by PCR amplificationusing the degenerate oligo-nucleotide primers5'TG(CT)AA(CT)AA(AG)AA(CT)GG(ACGT)TG(CT) and5'GC(CT)TT(AG)TTCCA(ACGT)CC(ACGT)GC(ACGT)GC(ACGT)CC and C. piscicolaJG126 genomic DNA as template. The 107 bp PCR product was cloned intothe pGEM®-T vector in the E.coli host PMC112 and the nucleotide sequenceof the cloned fragment determined. Translation of the sequence confirmedthat part of the piscicolin 126 gene had been cloned.

The 107 bp PCR fragment was used to screen the genomic lambda libraryand a positive clone containing an insert of approximately 20 kb wasisolated. From the lambda clone a 607 bp Hinf I/Sau 3A fragment wassubcloned into pGEf®-7 in the E.coli host PMC112 and the nucleotidesequence of both strands determined (FIG. 5). Translation of the DNAsequence confirmed that the gene encoding the piscicolin 126 and itsputative signal peptide had been cloned (FIG. 6).

Example 6

Spectrum of Antimicrobial Activity of Piscicolin 126

The spectrum of the antimicrobial -activity of piscicolin 126 has beentested against a number of pathogenic and non pathogenic bacteria andyeast strains.

The spectrum was determined in at least two separate tests againstGram-positive and Gram-negative bacteria and yeasts. Where a variableresult occurred in the first two tests, the indicator reaction wastested a third time and the result used to determine an overall positiveor negative reaction. The results are presented in Table 2.

Table 2. Antimicrobial Activity of Piscicolin 126.

Aliquots (5 μl) of a preparation of piscicolin 126 concentrated byammonium sulphate precipitation were applied to the surface of agarplates and allowed to dry prior to overlaying with indicator cultures.After incubation, the inhibition of the indicator strain by piscicolin126 was scored by the presence (+) or absence (-) of a zone of growthinhibition in the culture lawn.

    ______________________________________                                        AFRICC                                                                        Accession                                                                     No      Indicator               Activity                                      ______________________________________                                        0110    Aeromonas hydrophila    -                                             0303    Bacillus cereus         -                                             0312    Bacillus polvmyxa       -                                             0315    Bacillus stearothermophilus                                                                           -                                             0316    Bacillus stearothermophilus var. calidolactis                                                         -                                             0320    Bacillus subtilis       -                                             0602    Brocothrix thermosphactum                                                                             -                                             0603    Brocothrix thermosphactum                                                                             -                                             0604    Brocothrix thermosphactum                                                                             -                                             0605    Brocothrix thermosphactum                                                                             +                                             0901    Clostridium botulinum type B                                                                          -                                             0914    Clostridium sporogenes  -                                             1001    Corynebacterium sp.     -                                             1105    Debaryomyces hansei     -                                             1201    Enterobacter aerogenes NCTC 10006                                                                     -                                             1301    Escherichia coli NCTC 8196                                                                            -                                             2001    Lactococcus diacetylactis                                                                             -                                             2002    Lactococcus cremoris    -                                             2010    Lactococcus lactis      -                                             2011    Lactococcus lactis      -                                             2101    Lactobacillus sake      +                                             2102    Lactobacillus plantarum -                                             2103    Lactobacillus curvatus  +                                             2104    Lactobacillus sp.       -                                             2201    Leuconostoc mesentroides var. cremoris                                                                +                                             2206    Leuconostoc cremoris    -                                             2208    Leuconostdc dextranicus +                                             2301    Listeria denitrificans ATCC 14570                                                                     -                                             2303    Listeria grayi          +                                             2305    Listeria innocua        +                                             2306    Listeria ivanovii       +                                             2310    Listeria monocvtogenes 4A                                                                             +                                             2311    Listeria monocytogenes 4B                                                                             +                                             2312    Listeria monocytogenes  +                                             2320    Listeria seeligeri      +                                             2321    Listeria seeligeri      +                                             2405    Micrococcus luteus      -                                             2406    Micrococcus luteus      -                                             2415    Micrococcus varians     -                                             2702    Pediococcus acidilactici                                                                              -                                             2703    Pediococcus acidilactici                                                                              +                                             2704    Pediococcus pentosaceus +                                             3020    Proteus vulgaris        -                                             3101    Pseudomonas aeruginosa  -                                             3105    Pseudomonas fluorescens -                                             3301    Saccharomvces cerevisiae                                                                              -                                             3410    Salmonella salford      -                                             3412    Salmonella typhimurium  -                                             3501    Serratia marcescens     -                                             3601    Staphylococcus aureus NCTC 6571                                                                       -                                             3602    Staphylococcus aureus   -                                             3603    Staphylococcus aureus   -                                             3605    Staphylococcus camosus  -                                             3609    Staphylococcus epidermidis NGTC 6513                                                                  -                                             3715    Streptococcus thermophilus                                                                            +                                             3716    Streptococcus thermophilus                                                                            +                                             3801    Yersinia enterocolitica -                                             3901    Enterococcus faecalis   +                                             3902    Enterococcus faeceum    +                                             4001    Carnobacterium sp.      +                                             ______________________________________                                    

The results indicate that the bacteriocin has a limited spectrum ofactivity, and appears to act against food-borne pathogens and spoilagemicroorganisms but should have limited effect against beneficialbacteria such as cheese starter culture strains used in food systems.

Example 7

Quantitation of antimicrobial activity of piscicolin 126 against cheesestarter bacteria.

The sensitivity of cheese starter bacteria to piscicolin 126 was testedin both artificial growth media and whole milk (Tables 3, 4 and 5).

Table 3. Minimum Inhibitory Concentration of piscicolin 126 testedagainst cheese starter bacteria.

A preparation of piscicolin 126 concentrated by ammonium sulphateprecipitation was serially diluted in sterile distilled water andapplied (5 μl) to the surface of MRS agar plates and allowed to dryprior to overlaying with indicator cultures. The amount of bacteriocinJG 126 activity in the concentrated preparation was determined as204,800 AU ml⁻¹ by bioassay with Listeria monocytogenes 4A. Afterincubation. the minimum inhibitory concentration of piscicolin 126against each strain of cheese starter bacteria was expressed as thelowest amount of activity of piscicolin 126 which demonstrated adistinct zone of growth inhibition in the culture lawn.

    ______________________________________                                        Starter                                                                       Culture                                                                       Accession                     MIC                                             No.     Indicator             (AU ml.sup.-1)                                  ______________________________________                                        Mauri Laborataries Culture Collection (MLCC)                                  M124    Lactococcus lactis subsp. cremoris                                                                  >204,800                                        M126    Lactococcus lactis subsp. cremoris                                                                  51,200                                          M145    Lactococcus lactis subsp. cremoris                                                                  >204,800                                        M149    Lactococcus lactis subsp. cremoris                                                                  >204,800                                        M193    Lactococcus lactis subsp. cremoris                                                                  >204,800                                        M379    Lactococcus lactis subsp. cremoris                                                                  >204,800                                        M229    Lactococcus lactis subsp. lactis                                                                    >204,800                                        M238    Lactococcus lactis subsp. lactis                                                                    >204,800                                        M262    Lactococpus lactis subsp. lactis                                                                    >204,800                                        M276    Lactococcus lactis subsp. lactis                                                                    >204,800                                        M392    Lactococcus lactis subsp. lactis                                                                    >204,800                                        M474    Lactococcus lactis subsp. lactis                                                                    >204,800                                        DRC     Lactococcus lactis subsp. lactis var                                                                >204,800                                                diacetyladis                                                          FDLD3   Lactococcus lactis subsp. lactis var                                                                >204,800                                                diacetylactis                                                         HBD1    Lactococcus lactis subsp. lactis var                                                                >204,800                                                diacetylactis                                                         HDD1    Lactococcus lactis subsp. lactis var                                                                >204,800                                                diacetylactis                                                         FLIL1   Leuconostoc sp.       >204,800                                        HLD1    Leuconostoc sp.       >204,800                                        ST3     Streptococcus thermophilus                                                                          >204,800                                        ST41    Streptococcus thermophilus                                                                          >204,800                                        ST42    Streptococcus thermophilus                                                                          >204,800                                        AFRI Culture Collection (AFRICC)                                              2201    Leuconostoc mesentroides var. cremoris                                                              102,400                                         2208    Leuconostoc dextranicus                                                                             102,400                                         3715    Streptococcus thermophilus TS1                                                                      2,048                                           3716    Streptococcus thermophilus TS2                                                                      3,200                                           ______________________________________                                    

The results indicate that, in plate bioassays with Listeriamonocytogenes 4A, cheese starter bacteria are generally not sensitive toless than 2,048 AU ml⁻¹ of piscicolin 126 and most are not sensitive to102,400 AU ml⁻¹ and higher concentrations.

Table 4. The effect of Piscicolin 126 on cheese starter culture activityin whole milk.

The effect of piscicolin 126 (2,048 AU ml⁻¹) on starter culture activity(rate of pH decrease) was determined in sterile whole milk at 30° C.(except for strains 3715 and 3716, where an incubation temperature of37° C. was required to support more optimal growth). Cultures were grownin sterile skim milk (10% w/v) for 16 h at 30° C. or 37° C. (strains3715 and 3716) and inoculated (10% v/v) into sterile whole milk to teststarter activity in the absence and presence (2,048 AU ml⁻¹) ofpiscicolin 126. Uninoculated milk samples with and without addedpiscicolin 126 were used as controls. The pH of aliquots (2.5 ml) ofsamples removed under sterile conditions were determined at the timesindicated.

    ______________________________________                                        Starter culture pH                                                            Accession No.                                                                            JG126    0 h    2 h  4 h   6 h  8.5 h                              ______________________________________                                        MLCC M276  -        5.92   5.70 5.14  4.53 4.27                               (Bacteriocin                                                                             +        5.87   5.66 5.12  4.50 4.28                               insensitive)                                                                  MLCC M126  -        6.00   5.72 5.15  4.54 4.25                               (Bacteriocin                                                                             +        6.04   5.76 5.27  4.64 4.30                               sensitive)                                                                    AFRICC 3715                                                                              -        5.89   5.75 5.47  5.22 5.04                               (Bacteriocin                                                                             +        5.87   5.76 5.44  5.18 5.01                               sensitive)                                                                    ______________________________________                                    

The results indicate that piscicolin 126 at a level of 2,048 AU ml⁻¹ (asdetermined by plate bioassay with Listeria monocytogenes 4A) does notinhibit starter activity.

Table 5. The effect of piscicolin 126 on viable count of cheese startercultures and Listeria monocytogenes 4A in whole milk.

The effect of piscicolin 126 (2,048 AU ml⁻¹) on viable count of the mostbacteriocin-sensitive starter culture from each of the MauriLaboratories (strain M126) and AFRI (strain 3715) culture collections(Table 3) and Listeria monocytogenes 4A (at initial levels of 10⁴ and10⁶ cfu ml⁻¹) was determined in sterile whole milk (at 30° C. forstrains M126 and 2310 and at 37° C. for strain 3715) at the timesindicated. Viable counts were determined (as 0.1 ml spread plates of anappropriate dilution of the cultures) on M17 Agar (strains M126 and3715) and Listeria Selective Agar (strain 2310).

    ______________________________________                                                                Viable count                                                                  (log cfu ml.sup.-1)                                            Culture     JG126    0 h    6 h                                      ______________________________________                                        Starter cultures                                                                         MLCC M126     -        6.71 8.51                                   (on M17 Agar)            +        6.63 8.52                                              AFRICC 3715   -        7.15 8.08                                                            +        7.18 8.00                                   Listeria   inoculum 10.sup.4 cfu ml.sup.-1                                                             -        4.38 5.95                                   monocytogenes 4A         +        <1.0 <1.0                                   AFRICC 2310                                                                              inoculum 10.sup.6 cfu ml.sup.-1                                                             -        6.24 7.72                                   (on Listeria             +        <1.0 2.94                                   Selective Agar)                                                               ______________________________________                                    

The results indicate that the viable counts of cheese starter cultureswere unaffected, whereas the viable count of Listeria monocytogenes wassubstantially reduced by the presence of piscicolin 126 in whole milk.

The piscicolin 126 therefore should find application as a preservativefor foods and beverages.

Example 8

Antimicrobial Activity of Piscicolin 126 in Devilled Ham Paste andRicotta Cheese

Ricotta cheese (250 g) and Devilled Ham Paste (260 g) were inoculatedwith Listeria monocytogenes (AFRICC No. 2310) at 10³ -10⁶ cfu g⁻¹ insterile stomacher bags. An aliquot (10 ml) of concentrated piscicolin126 preparation was added and mixed thoroughly (Colworth stomacher, 60s) to provide a final concentration of piscicolin 126 of approximately2,048 AU g⁻¹ (as determined by plate bioassay). In control samples,sterile water was substituted for the piscicolin 126 preparation.Portions (10 g) of the mixtures were sealed in sterile stomacher bagsand incubated at 10° C. for the length of the trial. At the timesindicated. aliquots (90 ml) of peptone solution (0.1% w/v, pH 7) wereadded to portions of Ricotta cheese and mixed (Colworth stomacher, 60s). Viable counts were determined on aliquots (0.1 ml) of appropriatedilutions of the mixed samples (in peptone solution) spread onto thesurface of Listeria Selective Agar (Oxoid) plates. Viable countdeterminations were performed on each of three portions of Ricottacheese and Devilled Ham Paste at the times indicated and are expressedas the mean value in Tables 6 and 7.

Table 6. Effect of Piscicolin 126 on Listeria monocytogenes 4A inChallenged Ricotta Cheese.

The effect of piscicolin 126 (2,048 AU g⁻¹) on the viable count ofListeria monocytogenes 4A (initial inoculum 10⁴ cfu g⁻¹) in Ricottacheese spread was determined on Listeria Selective Agar (Oxoid) platesas the mean value of triplicate samples after incubation at 10° C., atthe times indicated.

    ______________________________________                                               Viable count on Listeria Selective Agar (Log cfu g.sup.-1)             Treatment                                                                              Day 0   Day 1   Day 2 Day 5 Day 7 Day 21                             ______________________________________                                        Control  4.49    4.85    5.57  7.20  7.67  8.26                               Piscicolin 126                                                                         2.12    2.00    2.52  4.08  4.95  7.87                               ______________________________________                                    

Table 7. Effect of Piscicolin 126 on Listeria monocytogenes 4A inChallenged Devilled Ham Paste.

The effect of piscicolin 126 (2,048 AU g⁻¹) on the viable count ofListeria monocytogenes 4A (initial inoculum 10³ cfu g⁻¹) in devilled hampaste was determined on Listeria Selective Agar (Oxoid) plates as themean value of triplicate samples after incubation at 10° C., at thetimes indicated.

    ______________________________________                                               Viable count on Listeria Selective Agar (Log cfu g.sup.-1)                      Day    Day     Day  Day   Day  Day   Day                             Treatment                                                                              0      1       4    6     7    14    21                              ______________________________________                                        Control  3.26   3.16    5.10 6.05  6.87 9.49  9.76                            Piscicolin 126                                                                         <2.0   <2.0    <2.0 <2.0  <2.0 <2.0  5.83                            ______________________________________                                    

The results indicate that under the experimental conditions growth ofListeria monocytogenes in Devilled Ham Paste is prevented by piscicolin126 for between 14 and 21 days, while growth of Listeria monocytogenesin Ricotta cheese is inhibited for up to 7 days.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 8                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "Primer"                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 19               CAG                                                        - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "Primer"                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "Primer"                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              26  GGAC GTTGCT                                                - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "Primer"                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #      37          TCCA CGTGCACGTG CACGTCC                                    - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 44 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: N-terminal                                          -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Carnobacteri - #um piscicola                                    (B) STRAIN: JG126                                                   #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Lys Tyr Tyr Gly Asn Gly Val Ser Cys Asn Ly - #s Asn Gly Cys Thr Val         #                15                                                           - Asp Trp Ser Lys Ala Ile Gly Ile Ile Gly As - #n Asn Ala Ala Ala Asn         #            30                                                               - Leu Thr Thr Gly Gly Ala Ala Gly Trp Asn Ly - #s Gly                         #        40                                                                   - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 607 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Carnobacteri - #um piscicola                                    (B) STRAIN: JG126                                                   #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - CGATGTTACA ATCAATTAAC TTTATAAGTT CATGAATAAT ATCGTGATAG TT - #CAGGAATA         60                                                                          - AAAAATCTAT AAGTAAAAAA GATGTGATAC AGTCAGCATG TTGTAAAAAA TA - #TTTTAAAA        120                                                                          - AGGAGCGTGT TTACGCATGA AAACTGTTAA AGAACTTAGC GTTAAAGAAA TG - #CAACTAAC        180                                                                          - TACAGGAGGT AAGTATTACG GAAATGGCGT TTCCTGTAAT AAAAATGGTT GT - #ACTGTAGA        240                                                                          - TTGGAGCAAA GCTATTGGGA TTATAGGAAA CAATGCAGCA GCAAATTTGA CT - #ACAGGTGG        300                                                                          - AGCCGCTGGT TGGAACAAAG GATAATTAAA GTCTCTTATT TTTTATCTTG TA - #AAAAAGAT        360                                                                          - GATACGCATC AATGCTGTGA CATAACATAG ATGGGTCTTT ATATTTGTAA GT - #TACATTTA        420                                                                          - AAACAAAAAT AAATATATAA AAATATTTTT TTATAGTCTT AGGAATTATG TT - #ATACTAAC        480                                                                          - AAAAATAGGC TAGTTTCAAC ATGATGTAAA GAAACTTATA CTATCAACTA AA - #ATCATAAA        540                                                                          - TATATAAAAT TAAGGAGTGA TATTTTATGG GTAAGTTAAA ATGGTTTTCT GG - #AGGAAAAG        600                                                                          #         607                                                                 - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 189 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Carnobacteri - #um piscicola                                    (B) STRAIN: JG126                                                   #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATGAAAACTG TTAAAGAACT TAGCGTTAAA GAAATGCAAC TAACTACAGG AG - #GTAAGTAT         60                                                                          - TACGGAAATG GCGTTTCCTG TAATAAAAAT GGTTGTACTG TAGATTGGAG CA - #AAGCTATT        120                                                                          - GGGATTATAG GAAACAATGC AGCAGCAAAT TTGACTACAG GTGGAGCCGC TG - #GTTGGAAC        180                                                                          #        189                                                                  - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: N-terminal                                          -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Carnobacteri - #um piscicola                                    (B) STRAIN: JG126                                                   #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Lys Thr Val Lys Glu Leu Ser Val Lys Gl - #u Met Gln Leu Thr Thr         #                15                                                           - Gly Gly Lys Tyr Tyr Gly Asn Gly Val Ser Cy - #s Asn Lys Asn Gly Cys         #            30                                                               - Thr Val Asp Trp Ser Lys Ala Ile Gly Ile Il - #e Gly Asn Asn Ala Ala         #        45                                                                   - Ala Asn Leu Thr Thr Gly Gly Ala Ala Gly Tr - #p Asn Lys Gly                 #    60                                                                       __________________________________________________________________________

What is claimed is:
 1. A substantially pure bacteriocin in which thebacteriocin has an amino acid sequence as shown in SEQ ID NO:5.
 2. Amethod of preserving foods or beverages, the method comprising adding tothe food or beverage an effective antimicrobial amount of a bacteriocinas claimed in claim
 1. 3. A composition of matter comprising a cheesestarter culture and a bacteriocin as claimed in claim 1.